A protocol for combining fluorescent proteins with histological stains for diverse cell wall components

• DOI: 10.1111/tpj.13784
• Publication Date: 2017-11-24T16:26:43Z
• Authors: Robertas Ursache; Tonni Grube Andersen; Peter Marhavý; Niko Geldner
• Keywords: Fluorescent protein

Higher plant function is contingent upon the complex three-dimensional (3D) architecture of tissues, yet severe light scattering renders deep, 3D tissue imaging very problematic. Although efforts to 'clear' tissues have been ongoing for over a century, many innovations made in recent years. Among them, protocol called ClearSee efficiently clears and diminishes chlorophyll autofluorescence while maintaining fluorescent proteins - thereby allowing analysis gene expression protein localisation cleared samples. To further increase usefulness this protocol, we developed ClearSee-based toolbox which number classical histological stains lignin, suberin other cell wall components can be used conjunction with reporter lines. We found that dyes are highly soluble solution, old staining protocols enormously simplified; these additionally unsuitable co-visualisation markers due harsh fixation clearing. Consecutive several allows distinct modifications as transcriptional reporters or tools deep within tissues. Moreover, easily applied on hand sections different organs. In combination confocal microscopy, improves image quality decreasing time cost embedding/sectioning. It thus provides low-cost, efficient method studying thick usually cumbersome visualise. Our ClearSee-adapted significantly improve speed up anatomical developmental investigations numerous species, hope they will contribute new discoveries areas research.